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Protein carboxymethylase of mouse pancreas is both soluble (70%) and particulate (30%). The Km for S-adenosylmethionine is 7.5 x 10(-7) M and the Ki for S-adenosylethionine is 1.3 . 10(-5) M. Administration of an ethionine containing diet results in a decrease in protein carboxymethylase activity. Ethionine ingestion also increases pancreatic amylase content by interfering with digestive enzyme discharge. The reciprocal changes in amylase content and protein carboxymethylase activity can be detected within 12 h of commencing the ethionine administration and are enhanced by simultaneous choline deficiency. These studies support the hypothesis that protein carboxymethylase plays an important role in secretion of exportable material. Inhibition of pancreatic protein carboxymethylase activity in vivo may be one important mechanism by which ethionine interferes with digestive enzyme discharge.

Induction of hepatic steatosis and suppression of hepatic ATP levels, protein synthesis and gluconeogenesis subsequent to administration of ethionine may be consequences of interference by this compound with mitochondrial phosphorylation of ADP. The mitochondrial dysfunction is not a direct action of ethionine on the organelle.

Sheep were given intravenous infusions of ethionine together with cycloleucine or reduced glutathione, in attempts to prevent the inhibition of wool growth by ethionine. Other sheep were given cycloleucine alone to measure effects on wool growth. Twenty-two compounds related to cystine, methionine, ethionine, lysine, phenylalanine and tyrosine were given as intravenous infusions to sheep to investigate their potential as depilatory agents. Nineteen of these compounds were also tested in mice during their first cycle of hair growth. The concurrent administration of cycloleucine with ethionine prevented the weakening of wool fibres caused by ethionine, but reduced glutathione was ineffective. Cycloleucine weakened wool fibres, as judged subjectively, and caused a small reduction in fibre diameter. Selenocystine and selenomethionine caused some hair loss in mice but selenocystine was also toxic. Both seleno-amino acids were toxic for sheep; selenocystine was lethal at 0.025 mmol kg-0.75 and selenomethionine at 0.09 mmol kg-0.75. Doses that permitted survival of sheep did not have depilatory effects. However, the presence of autophagic vacuoles in the cytoplasm of follicle bulb cells of sheep indicated that a toxic dose of selenocystine had potential depilatory activity. Other compounds investigated did not induce loss of wool or hair. Some compounds, notably 3-methylthiopropionic acid and S-(2-aminoethyl)-L-cysteine, were toxic to mice but not sheep. The methionine analogue, methoxinine (O-methyl-DL-homoserine), caused a substantial reduction in the strength of wool fibres and a prolonged alteration of the crimp pattern. It is suggested tentatively that cycloleucine inhibits methionine adenosyltransferase and thereby reduces or prevents the formation of S-adenosylethionine. The failure of various compounds related to methionine and ethionine to have any depilatory activity in sheep supports the view that ethionine influences wool growth via the formation of S-adenosylethionine.

The influence of the cellular level of adenosine triphosphate (ATP) in the liver on bile excretion was studied in rats. In ischemia, the cellular ATP level decreased rapidly--and, concomitantly, bile flow stopped within 5 min. Administration of L-ethionine i.p. to rats reduced the bile flow rate with decrease in the cellular ATP level. The correlation between the bile flow rate and the cellular ATP level was confirmed in a liver perfusion system. On anoxic perfusion, the ATP level and bile flow rate changed in the same manner as in ischemia. The recovery rates of both on reoxygenation decreased with increase in the anoxic perfusion period. During perfusion under oxygenated conditions, decrease in cellular ATP to various levels by infusion of various concentrations of potassium cyanide, an inhibitor of respiration, resulted in corresponding and concomitant suppression of bile excretion. Kinetic analysis of the bile flow rate revealed a Michaelis-Menten-type curve for the cellular ATP level. The apparent Kms for ATP of bile flow rate in L-ethionine-treated rat liver and liver perfused with potassium cyanide were 1.0 and 1.6 mM, and their Vmax values were 4.1 and 2.5 microliter/min/g liver, respectively. The concentrations of main bile components, such as phospholipids, cholesterol, and taurocholate increased, but their total outputs decreased with decrease in the ATP level, and returned to the normal range with recovery of the ATP level. Thus, it was shown experimentally that the extent of hepatic injury can be assessed simply by monitoring the bile flow rate, which reflects the cellular level of ATP.

The extensional growth rate of wild-type 74A8 N. crassa in the presence of various concentrations of 19 amino acid analogs was measured. Seven analogs were not inhibitory at concentrations in the range of one to 10mM. Of the remaining 12 analogs, nine inhibited growth in a novel way. The kinetics of growth in the presence of these analogs at 30 degrees were characterized by seven sequential phases: (1)lag; (2) acceleration of growth rate; (3) steady-state growth rate; (4) exponential rate of decline of growth rate; (5) no growth or growth rate less than or equal to 0.1 mm h-1; (6) accleration of growth rate; and (7) steady state. At 33 degrees, phases 6 and 7 did not occur and irreparable death of the clones occurred. The mechanism by which the clones acquired resistance at 30 degrees appeared to involve a combination of physiological adaptation and cellular selection. Dietary application of either free radical scavengers or surface-active membrane 'stabilizers' alleviated or prevented the inhibition and deterioration of growth rate which occurred in the presence of the nine amino acid analogs. Culture with either 4-fluorophenylalanine or ethionine led to an increase of the activities of antioxygenic enzymes glutathione peroxidase, glutathione reductase and superoxide dismutase. The amino acid analogs that cause senescence and death of growing cells are known to be incorporated into proteins and such proteins are generally abnormal. Because a substantial fraction of cellular protein occurs in membranes and the proteins synthesized by mitochondria are exclusively intrinsic membrane proteins, we suggest that a primary consequence of errors in protein synthesis is the production of faulty membranes. The deterioration of such membranes with associated lipid autoxidation and free radical production proceeding as a chain reaction at an exponential rate may in itself contribute to the exponential rate of cellular deterioration which is characteristic of the ageing process. According to this hypothesis, dietary membrane stabilizers, free radical scavengers and antioxygenic enzymes protect cells from error catastrophy arising from the chain of events leading from membrane deterioration.

Mouse lymphoma cells were shown to be unresponsive to prostaglandin E1 in terms of cAMP production. The endogenous ATP concentration was shown to be low. Addition of ATP in the presence of IMBX into the incubation medium resulted in elevation of the intracellular pool of ATP and the cells responded to PGE1 by increasing cAMP production. The ATP effect is specific and cannot be substituted by GTP. ATP analogue (AMP-PCP) can, however, produce a similar effect to ATP. The intracellular ATP concentration can be lowered by incubation with iodoacetate and potassium cyanide. This caused a drastic decrease of the cAMP level. Ethionine on the other hand had no effect on the intracellular ATP level.

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

Formation of sporangia can be induced in starved macroplasmodia of Physarum polycephalum by illumination. This process of morphogenesis which starts 9 h after the needed period of illumination can be prevented by the competitive inhibitors of methyltransferases S-adenosyl-homocysteine or L-ethionine, applied either directly by microinjection into the plasmodia or by addition to the medium. Because 5-azacytidine (aza-C) or 5-aza-2'-deoxycytidine (aza-dC) also prevent sporulation (in contrast to cytidine, 8-azaguanine (aza-G) or 6-aza-uridine (aza-U) it is suggested that DNA is the substrate for methylation. The injection technique allows one to determine the period of methylation (i.e. between the 3rd and 4th h of the cell differentiation process after the induction by illumination). Based on the correlation between methylation and genome expression, it is suggested that some genes must be repressed by methylation during this period.

Histological and ultrastructural aspects of acute and chronic pre-erythrocytic schizonts of P. yoelii yoelii were studied with: a) acute schizonts in normal rodents; b) chronic schizonts induced in rodents receiving ethionine injections or on a low methionine diet; Experimentally induced chronic schizonts frequently have the same histological aspect as those observed in wild Thamnomys from Africa. The ultrastructural evolution of the vacuole system described by Seureau et al. in acute schizonts is discussed; new evidence is presented to support the hypothesis according to which the contents of the vacuoles are enzymatic in nature. Ultrastructural studies of chronic schizonts show alterations in the sites of synthesis (zones of rough endoplasmic reticulum small and not numerous, absent in the most delayed schizonts; vesicle system poorly developed and also disturbances of nuclear development (slow division, apparently inhibited in the most retarded forms.) A peripheral accumulation of vacuoles in many chronic schizonts without discharge of their contents to the parasitophorous space, might indicate an alteration of their membranes, leaving them unable to fuse with the schizont plasmalemma.

In a previous report, administration of [3H]ethylethionine to partially hepatectomized rats was shown to ethylate two classes of proteins extracted from rat liver nuclei in 0.25 N HCl. Upon acid hydrolysis of the proteins, ethyl derivatives of both lysine and arginine were found. The arginine derivative which represented the major ethylated product is identified in this report as NG-monoethylarginine by the use of alkali hydrolysis. Administration of methionine along with the ethionine partially inhibited the ethylation. This suggests that the ethylation may proceed in a similar manner to normal protein methylation by which the methylases utilize S-adenosylmethionine as the ethyl donor. Using in vitro assays for both protein-lysine and protein-arginine methyltransferase it was found that only the protein-arginine methyltransferase could use radiolabeled S-adenosylmethionine as a substrate. The major product formed in this assay was identified as NG-monoethylarginine.

One hundred eleven male adult rats of a pathogen-free Sherman or Charles River strain were divided into 3 series. Each of them contained non-treated controls and rats treated with intraperitoneal (i.p.) injections of DL-ethionine (E) [75 mg/d p. 100 g] for 6 weeks (series 1) or for only 2 weeks (series 2 and 3). Series 1 and 2 included also rats injected simultaneously with E and DL-methionine (68.4 mg/d p. 100 g). In the series 2 there were also groups receiving at the same time E, ME and tryptophan (5 mg/d p. 100 g) or E, Me and ATP (1.65 mg/d), and in the series 3, groups were injected with E + adenosine (31.2 mg/d p. 100 g) and (or) uridine (284 mg/d p. 100 g). All animals were immunized i.p. 1 week before killing with sheep red blood cells (SRBC): either 2.4 X 10(9) SRBC p. 100 g without adjuvant (series 1 and 2) or 1 X 10(9) SRBC with Freund's adjuvant added (series 3). Rosette-and plaque-forming cells (RFC and PFC) were counted in the spleen, and titers of serum hemagglutinins and hemolysins were determined with separation of 2-mercaptoethanol-resistant IgG from non-resistant immunoglobulins. E provoked within 2 weeks a drastic inhibition of the immune responses: drop in the RFC and PFC levels p. 10(6) (approximately 2900 leads to approximately 320 and approximately 415 leads to 40 respectively, in the study 3), and significant decrease in the serum antibody titer, especially IgG. Addition of Me still amplified the immunosuppression. Supplementation with tryptophan, ATP, adenosine and (or) uridine was also ineffective. Though not displaying any immunoprotective potency, Me partly neutralized the non-immunological effects of E: arrest of the body growth and involution of lymphoid organs and male genital organs, particularly apparent after 6 weeks of treatment. In conclusion, the ethionine-induced immunosuppression does not result from a metabolic exclusion of labile methyl groups or from an acute ATP deficiency due to an excess trapping of adenine as S-adenosyl-ethionine. Lack of pyrimidines or tryptophan must also be discarded. On the other hand, the possibility of maintaining the immunosuppressive activity of E, while reducing the toxicity of this compound, by addition of Me deserves to be emphasized.

The dose-dependent and sex-related effects of 3 hepatocarcinogens were investigated by measuring the number and area of gamma-glutamyl transpeptidase (gamma-GT) positive foci appearing in the liver. For this, three different doses of 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and DL-ethionine (ethionine), respectively, were given to F344 rats of both sexes for 6 weeks after a single injection of diethylnitrosamine (DEN). The inductions of gamma-GT positive foci by 2-AAF and 3'-Me-DAB were clearly dose-dependent, but with 2-AAF this was clearer in males than females. Ethionine had less clear dose-dependent effects in both males and females. Under these conditions, the minimal effective doses of 2-AAF, 3'-Me-DAB and ethionine, respectively, were 0.0008%, 0.0024% and 0.05% in males and 0.004%, 0.012% and 0.05% in females. The results indicate that these 3 carcinogens had clear dose-dependent effects in the induction of gamma-GT positive foci when given for a short period in the promotive stage and that male rats were more susceptible than females to 2-AAF.

Nuclear protein methylation was studied in regenerating rat liver by giving [methyl-3H]methionine 45 h after partial hepatectomy. Ethionine, a liver carcinogen, has been shown to alter the methylation patterns in a basic protein (histone) fraction, as well as an acidic protein (non-histone) fraction present in a 0.25 N HCl nuclear extraction. The proteins present in the 0.25 N HCl extraction were separated by chromatography using a Bio-Rex 70 cation exchange column. Polyacrylamide gel electrophoresis and total amino acid analysis showed the first protein fraction contained acidic large molecular weight non-histone proteins, while the second fraction contained basic small molecular weight histone proteins. Both fractions were then hydrolyzed, and the amino acids chromatographed on an Aminex A-5 cation exchange column. The histones were found to contain epsilon-N-mono, di and trimethyllysine derivatives; whereas the non-histone fraction contained these lysine derivatives and additional basic amino acid identified as NG,NG-dimethylarginine. Ethionine (0.5 mg/g body weight) was found to inhibit in vivo methylation of lysine to form epsilon-N-mono, di and trimethyllysine, 46, 52 and 68%, respectively. The formation of NG,NG-dimethylarginine was inhibited by 85%. Ethylation of these proteins was also studied by giving [ethyl-3H]ethionine. After hydrolysis, the non-histones were found to contain a labeled lysine and arginine derivative, but in the histone fraction only labeled lysine was found.

As a direct consequence of theoretical considerations of carcinogen-induced embryonic gene activity, it now appears possible that, in principle, economically important embryonic plant genes can be activated artificially and thereby induce major increases in yields of products such as starch in grain endosperm. Furthermore it should be noted that this theory of genetic manipulation of genes has no counterpart in present genetic engineering. The technique of gene splicing, etc. introduces new genetic elements or rearrangements, whereas the method proposed here does not require changing the genome - only its expression. This idea should have great implications for agro-industry and world food production in general.

In Escherichia coli, DNA damage by carcinogenic agents results in the coordinate expression of a diversity of functions (SOS functions), many of which are thermally inducible without any damage to DNA in a tif-1 mutant. These include prophage induction, filamentous growth, and an error-prone DNA repair activity, which is responsible for ultraviolet-induced mutagenesis. Ethionine causes hepatic carcinoma in rats after prolonged feeding but is not a mutagen in the Ames test. The present study shows that 10 mM ethionine prevents the thermal induction of lambda-prophage in a tif-1 derivative of E. coli. The enhancement of mutation, which normally occurs at high temperature after a low dose of ultraviolet light, is also blocked by ethionine. Ethionine does not block, to any appreciable extent, the incorporation of radioactive precursors into RNA, DNA, or protein.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.

The cytotoxic response of thymocytes to chemical inducers of differentiation does not represent a non-specific toxic action of these drugs. The death of thymocytes treated with the inducers or with gamma-rays is associated with internucleosomal chromatin fragmentation. All treatments are more effective on the most radiosensitive sub-population of these cells. This subpopulation is characterized by the maximal level of spontaneous DNA lesions. Incubation of thymocytes with the inducers of differentiation raises the level of these lesions. It is suggested that the processes of thymocyte death after the inducer treatment or irradiation and of cellular differentiation have features in common, and the capacity of thymocytes to limit or reverse the potentially lethal effects of these treatments is determined by the level of pre-existing spontaneous DNA lesions.

Expression of the glycogenolytic action of glucagon in liver requires ATP for cAMP formation and for several subsequent phosphorylation reactions. To assess the extent to which ATP availability is rate-limiting to this hormonal action, responses to glucagon of intact liver and of liver with marked reductions in ATP content induced by ethionine was examined in female Wistar rats in vivo and in vitro. Compared to values in quick-frozen liver samples from control rats, basal hepatic ATP was 75% lower and cAMP, two fold higher in rats treated with ethionine. Activation of glycogen phosphorylase and inactivation of glycogen synthetase, phosphorylation reactions which require ATP and are initiated by cAMP, were also evident in basal liver samples from ethionine-treated rats. These hepatic alterations were associated with portal glucose and insulin levels which were significantly lower and portal glucagon levels which were four fold higher than values in controls. In ethionine-treated rats, glucose infusion decreased hepatic cAMP content and phosphorylase activity and increased synthetase activity. This and other observation suggested that the higher cAMP and the altered enzyme activities seen in vivo after ethionine administration were mediated by the hyperglucagonemia and/or by other endogenous glycogenolytic stimuli, and accordingly implied that liver remained responsive to such stimuli despite reduced ATP. Pharmacologic doses of exogenous glucagon clearly increased cAMP in vivo and in vitro in livers with decreased ATP. However, the lower ATP of liver exposed to ethionine was associated with a significantly blunted cAMP response to maximal glucagon stimulation. By contrast, alterations in phosphorylase and synthetase activities were not similarly blunted, suggesting that the smaller increases in cAMP seen in liver with reduced ATP content were adequate for the expression of these actions of the hormone. It is concluded that the actions of glucagon to increase cAMP and to activate phosphorylase and inactivate synthetase are not abolished by marked reductions in hepatic APT.

Simultaneous administration of cysteine and DL-ethionine in food protects, and enhances, respectively, the tissue damage produced by parenteral administration of cadmium (CdCl2) in testicles of rats. The effects are explained as an inactivation of SH-groups by cadmium, and in interference in cysteine metabolism by ethionine, respectively.

The biosynthesis of etamycin by Streptomyces griseoviridus was investigated with the precursors, 14C-L-leucine and 14CH3-L-methionine, during short-term incubations. Both radiosotopes are rapidly incorporated into the antibiotic without significant lag. D-Leucine,but not L-leucine,inhibits etamycin formation. The kinetics of incorporation of 14C-L-leucine as well as the inhibitor studies suggest that L-leucine is the direct precursor of theD-enantiomerpresent in the antibiotic peptide. Chloramephenicol has been shown to inhibit protein synthesis by S. griseoviridus without reduction of etamycin formation, indicating that a non-ribosomal mechanism of synthesis is involved in etamycin biogenesis. When L-ethionine was employed, both the formation of antibiotic and protein were blocked.

Treatment of rats with an ethionine plus adenine or a methionine diet leads not only to a marked increase of the alpha-form isozyme of S-adenosylmethionine synthetase in liver, but also to the accumulation of comparable amounts of S-adenosylethionine and S-adenosylmethionine in liver. Transplantation of ascites tumor cells into mice leads to a marked increase only of the beta-form isozyme in the host liver, but the levels of S-adenosylmethionine do not significantly change in liver.

A pleitropic ethionine-resistant mutant of Neurospora contains an S-adenosylmethionine synthetase that is labile to heat and dialysis but exhibits normal kinetics for methionine and ethionine.

S-Adenoxyl-L-methionine (SAM) inhibited leucine transport in Saccharomyces cerevisiae. By using a mutant defective in the active transport of SAM, we demonstrated that the inhibitory effect was exerted at an extracellular site. Cells preincubated wtih SAM for 120 min became refractory to its inhibitory effect, which was not a result of either the active transport or the metabolism of SAM. The quantitative recovery of labeled SAM from the incubation medium indicated that SAM, and not a metabolite, was the true inhibitory molecule. S-Adenosyl-L-homocysteine and S-adenosyl-L-ethionine also functioned as inhibitors of leucine transport, whereas S-adenosyl-D-methionine, S-adenosyl-D-homocystein, 5'-methylthioadenosine, 5'-dimethylthioadenosine, and adenosine lacked this property. Kinetic studies demonstrated that SAM was a competitive inhibitor of leucine transport.

The purpose of the present study was to elucidate the metabolic requirements of autophagocytosis. Two model systems were used for this purpose: a) glucagon-induced autophagocytosis in the rat liver, and b) the wave of autophagocytosis which occurs when isolated flounder kidney tubules are incubated in vitro. In the rat liver, protein synthesis was inhibited by the administration of cycloheximide (1.5 mg/kg) to rats 2 hours prior to glucagon injection. In flounder kidney tubules, protein synthesis was inhibited at least 90% by adding cycloheximide, actinomycin D, pactamycin and puromycin to the medium. In both systems the inhibition of protein synthesis failed to inhibit the formation of autophagic vacuoles or their subsequent transformation into autolysosomes, as depicted from electron microscopic histochemical preparations. In flounder kidney tubules no differences were found in the levels of p-nitrophenylphosphates, beta-DL-glycerophosphatase, N-acetyl-beta-D-glucosaminidase, arylsulphatase, beta-D-galactosidase or acid proteinase when tubules were incubated up to 5 hours in the presence or absence of protein synthesis inhibitors. When ethionine was administered to rats 2 hours before glucagon injection, a decrease of approximately 75% in the ATP levels was observed. After ethionine administration, glucagon failed to induce the formation of autophagic vacuoles. The incubation of flounder kidney tubules in the presence of cyanide or in a nitrogen atmosphere decreased the ATP levels to less than 10% of controls and blocked autophagy. On the other hand, cyanide had little effect on acid hydrolase levels at 1 hour of incubation. A wide variety of other inhibitors were also shown to block autophagy. These results further support the hypothesis that, in the formation of antophagic vacuoles, preexisting enzyme and membrane pools are utilized. On the other hand, the esotropy-exotropy membrane conformational changes occurring in the formation of autophagic vacuoles seem to be energy dependent and can therefore be blocked by lowering intracellular ATP levels.

Zoxazolamine paralysis, hexobarbital anesthesia and digitoxin convulsions were diminished in rats by 3 days of pretreatment with pregnenolone-16alpha-carbonitrile (PCN). Simultaneous injection of dl-ethionine did not block this effect of PCN. Dl-Ethionine decreased, while PCN or PCN + ethionene increased, zoxazolamine metabolism by the 9,000g supernatant fraction of the liver. Dl-ethionine neutralized the influence of PCN and phenobarbital on zoxazolamine metabolism in rats that were pretreated with PCN or phenobarbital for 1 day, and it inhibited the action of phenobarbital on hexobarbital anesthesia. PCN augmented the liver weight of animals given dl-ehtionine whereas dl-ethionine in itself did not. PCN produced SER proliferation in hepatocytes and dl-ethionine caused lipid accumulation, SER proliferation and myelin-figure formation. When administered conjointly, these compounds had an additive effect on the liver cell ultrastructure.

Treatment of mice with DL-ethionine and adenine causes a reduction of all methylated bases of liver tRNA. This effect is dose-dependent and specific for the methylated bases. Individual methylated components are affected to different extents, m22G being most sensitive to inhibition.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.

The effects of the administration of different fatty liver inducing drugs on the serum lipoprotein lipase activating ability was investigated in rats. Addition of serum from 2-mercaptoethanol-, 2-mercaptoacetate-, ethionine- or D-galactosamine- treated rats failed to activate heart and adipose tissue lipoprotein lipase from control rats. The activating effect of serum was only slightly reduced in isopropanol-treated rats, whereas it was found unaffected in ethanol-treated ones. Electrophoresis of the lipoproteins and of the very low density lipoproteins (VLDL) fraction of sera from 2-mercaptoethanol-, 2-mercaptoacetate-, isopropanol-, ethionine- and D-galactosamine-treated rats suggest that the lack of lipoprotein lipase activation ability of these sera is most probably related to the impairing effects of these drugs upon VLDL metabolism, i.e. reduction of VLDL secretion in the case of 2-mercaptoethanol, 2-mercaptoacetate and isopropanol, production of abnormal VLDL in the case of D-galactosamine and both decreased VLDL secretion and production of abnormal VLDL in the case of ethionine.